| Objective: Obesity-related hypertension refers to hypertension caused by various factors of obesity.Obesity and hypertension demonstrate strong synergistic effects.Body fat volume is positively correlated to blood pressure.Framingham Heart Study reported that 60-70% of adult essential hypertension is closely related to obesity.The coexistence of obesity and hypertension not only increases the difficulty of blood pressure control,but also aggravates metabolic and cardiovascular disorder and increases the risk of cardiovascular and cerebrovascular events.Interaction of multiple factors of the activation of renin angiotensin aldosterone system and sympathetic nervous system,leptin resistance,insulin resistance,sodium retention and inflammation/oxidative stress leads to the vicious circle of obesity and hypertension and promotes the progression of obesity-related hypertension.Oxidized low density lipoprotein(ox-LDL)induces endothelial injury and triggers arterial remodeling by regulating gene transcription,which plays a vital role in the development of obesity-related hypertension.From the perspective of traditional Chinese medicine(TCM),the main pathogenic factors of obesity-related hypertension are disturbance of wind and phlegm.The famous doctor,Dr.Zhu Dan Xi(1281-1358 AD)in Yuan Dynasty,has proposed the theory of "no phlegm,no dizziness" and "fat people always with phlegm and dampness".Banxia Baizhu Tianma Decoction(BBTD)is composed of Pinellia ternata(Ban Xia),Gastrodia(Tian Ma),Atractylodes macrocephala(Bai Zhu),Red tangerine peel(Ju Hong),Poria cocos(Fu Ling),Glycyrrhiza uralensis(Gan Cao),Zingiber(Sheng Jiang)and Jujube(Da Zao).BBTD is the classical representative prescription for expelling phlegm,extinguishing wind,strengthening the spleen and dissipating excessive fluid in TCM,which is a common used to treat hypertension and hypertension-related symptoms in clinical practice in China and many East Asian countries.Real world studies have confirmed that BBTD effectively alleviates dizziness and migraine and reduces the risk of stroke and dementia.In the present study,the murine model of hypertension with excessive accumulation of phlegm-dampness was induced by high-fat diet and used to explore the potential syndrome markers by the plasma non-targeted metabonomics assay from an overall perspective to explain the metabolic characteristics of hypertension with excessive accumulation of phlegm-dampness.Then,we focused on the effect of BBTD on the plasma metabolite profile of obesity-related hypertensive mice,and evaluated the protective effect of BBTD on ox-LDL induced endothelial injury according to the result of plasma metabonomics.And then the targets of BBTD on endothelium were explored through the integrated analysis of transcriptome sequencing and small RNA sequencing and si RNA knockdown/overexpression technology.Meanwhile,β-sitosterol,as one of the main monomer components of Pinellia ternata(principal drug of BBTD),was selected to evaluate its protective effects on ox-LDL induced endothelial injury and explore its targets in vitro,so as to authenticate the molecular basis of BBTD.Methods: 1.C57BL/6cnc mice were fed with high fat diet for 25 weeks to induce the murine model of hypertension with excessive accumulation of phlegm-dampness.The general status of mice and the tissue morphology of aorta were observed.The plasma non-targeted metabonomics were assayed by Ultra performance liquid chromatography/tandem mass spectrometry(UPLC-MS/MS).2.Obesity-related hypertensive mice were induced by high-fat diet for 20 weeks.BBTD(17.8 g/kg)was administered intragastrically for 8 weeks,and telmisartan(12.5mg/kg)was used as positive drug.Body weight,blood pressure,triglyceride and cholesterol were recorded to evaluate the efficacy of BBTD in vivo.Lipid deposition in aortic root was assessed by oil red O staining and morphology of aortas was observed by hematoxylin-eosin(HE)staining.UPLC-MS/MS was performed to study the plasma non-targeted metabonomics.According to the data of metabonomics,human aortic endothelial cells(HAECs)were cultured and treated by ox-LDL(50 μg/mL)with/without BBTD in different dosages(2,1 or 0.5 mg/mL).Apoptosis rate was determined by Annexin V-FITC/PI,migration was determined by Transwell,cytoskeleton was observed by Phalloidin staining and density of VE-cadherin was observed by immunofluorescence staining,to investigate the effects of BBTD comprehensively in vitro.3.Transcriptome Sequencing and Small RNA Sequencing was performed(2 mg/mL BBTD vs ox-LDL)to screen the possible targets of BBTD in endothelial protection against ox-LDL.Based on knockdown/overexpression of mi RNA-217 by si RNA,BBTD was added simultaneously to assess the expression of mi RNA-217 and its corresponding protein-coding transcripts,and subsequently its effect on cell proliferation was evaluated by MTT assay and the levels of IL-6,IL-1β,ICAM-1 and TNF-α in supernatant were evaluated by ELISA assay.4.HAECs were cultured in vitro and treated by oxidized low-density lipoprotein(ox-LDL,50 μg/mL)with/without β-sitosterol(2 μg/mL)for 24 h.Apoptosis rate was determined by Annexin V-FITC/PI,migration was determined by Transwell,the mitochondrial oxygen consumption rate(OCR)was assayed by a Seahorse XF(e)24Extracellular Flux Analyzer,cytoskeleton was staining by Phalloidin and density of VE-cadherin was observed by immunofluorescence staining,to investigate the effects ofβ-sitosterol in vitro.Transcriptome sequencing and Small RNA Sequencing was performed to screen the possible targets of β-sitosterol.Results: 1.Model mice demonstrated the characteristics of excessive accumulation of phlegm-dampness,the body weight,Lee’s index,systolic blood pressure,diastolic blood pressure,fasting insulin,blood glucose/insulin and leptin levels were increased(P < 0.05).Obvious morphological changes and lipid deposition in the aorta were observed in model mice.A totel of 125 differental metabolites(64 increased and 61 decreased;VIP > 1 and P< 0.05)were screened out by UPLC-MS assay.The most significant metabolites were phosphatidylcholine,lysophosphatidylcholine,L-isoleucine and acetylcarnitine.The differental metabolites were enriched in choline metabolism,Gn RH signaling pathway,amino acid biosynthesis and FCγR-mediated phagocytosis(P < 0.01),and thermogenesis,EGFR,NF-κB,c AMP and m TOR signaling pathway(P < 0.05).2.BBTD effectively reduced the body weight and total cholesterol,and decreased12.1 mm Hg in SBP and 10.5 mm Hg in DBP of obesity-related hypertensive mice(P <0.05).BBTD attenuated lipid deposition in arterial roots and improved the morphology of aortas in vivo.Plasma metabolite profiles identified 94 differential metabolites and suggested BBTD mainly affected glycerophospholipids and fatty acyls.Bioinformatics analysis indicated sphingolipid metabolism and fluid shear stress and atherosclerosis were main pathways.Therefore,we focused on thes effect of BBTD on endothelium against ox-LDL.In vitro,BBTD demonstrated endothelial protective effects,decreasing apoptosis rate,improving cell migration in dose-dependent manner and maintaining cell morphology.3.Transcriptome sequencing screened 759 differentially expressed(DE)mRNAs in HAECs after ox-LDL induction,of which 160 mRNAs were up-regulated and 559 mRNAs were down regulated(FC > 2.0,P < 0.05,ox-LDL vs untreated HAECs);After BBTD treatment for 24 h,251 mRNAs were down-regulated and 603 mRNAs were up-regulated(FC > 2.0,P < 0.05,BBTD vs ox-LDL),suggesting that BBTD reversed 51.8% of the mRNA change induced by ox-LDL(393 overlapped DE mRNAs).Bioinformatics analysis confirmed the effectiveness of BBTD in hypertension and suggested that BBTD improved endothelial injury mainly by targeting p53 and PPAR pathways.Small RNA sequencing showed that a total of 90 DE mi RNAs were screened in HAECs after ox-LDL induction.BBTD leaded to 157 mi RNAs differentially expressed(93 were up-regulated,64 were down regulated,FC > 2.0,P < 0.05),suggesting that BBTD reversed 66.67% of the mi RNA changes induced by ox LDL(60 overlaped DE mi RNAs),among which the most significantly different mi RNA was hsa-microRNA-217-5p(mi RNA-217).By knockdown of mi RNA-217,cell proliferation was inhibited and inflammatory cytokine levels of IL-6,IL-1β,ICAM-1 and TNF-α were increased;while overexpression of mi R-217 resulted in converse accordingly.The HAEC proliferation was decreased and levels of IL-6,IL-1β,ICAM-1 and TNF-α were increased,though exposure to BBTD after mi RNA-217 knockdown,suggesting the efficacy of BBTD was blocked by mi RNA-217 knockdown;while,overexpression of mi RNA-217 enhanced the proliferation and anti-inflammatory effects of BBTD.4.β-Sitosterol significantly inhibited cell apoptosis(P<0.01),increased cell migration(P < 0.01),improved energy metabolism(P < 0.05)and improved morphology after ox-LDL exposure following β-sitosterol(2 μg/mL)treatment in HAECs.A total of 691 DE mRNAs were identified(579 were upregulated and 112 were downregulated,FC ≥ 2.0,P <0.05)after 24 h of β-sitosterol administration in transcriptome sequencing(β-sitosterol vs ox-LDL),which suggested sitosterol reversed 62.32% change in mRNAs induced by ox-LDL.Mi RNA-seq data proposed 87 up-regulated and 58 down-regulated mi RNAs(FC≥ 2.0,P < 0.05)in mi RNA-seq(β-sitosterol vs ox-LDL),suggesting β-sitosterol reversed76.67% change in mi RNAs induced by ox-LDL.DE mi RNAs-DE mRNAs coexpression network focused on ribosome,cell cycle,oxidative phosphorylation,PI3K-Akt signaling pathway,TNF signaling pathway,Erb B signaling pathway and m TOR signaling pathway.Consequently,mi RNAs might be the targets of β-sitosterol and play vital roles in transcriptional regulation in endothelial protective effects against ox-LDL.At their respective optimum concentrations,both β-sitosterol and BBTD demonstrated convincing endothelial protective effects on apoptosis,migration,cytoskeleton and morphology.At the transcriptional level,there were 314 overlapped mRNAs(36.77% of the mRNA targets of BBTD);At the post transcriptional level,there are 75 overlapped mi RNAs(47.78% of the mi RNA targets of BBTD),suggesting that β-sitosterol is one of the main effective substances of BBTD for endothelial protection.Conclusion:1.Phosphatidylcholine,lysophosphatidylcholine,L-isoleucine and acetylcarnitine are suggested as the characteristic plasma metabolic biomarkers of hypertension with excessive accumulation of phlegm-dampness.2.BBTD modulates plasma metabolite profiles(glycerophospholipids and fatty acyls are the main affected metabolites)and endothelial protection to attenuate obesity-related hypertension in vivo.BBTD decreases apoptosis rate,improves migration and maintains cell morphology of HAECs in dose-dependent manner in vitro.After BBTD administration for 24 h,854 DE mRNAs and 157 DE mi RNAs were screened,reversing 51.8% of the mRNA changes and 66.67% of the mi RNAs changes induced by ox-LDL,among which mi RNA-217 was the one with the most significant difference.By si RNA knockdown/overexpression verified that BBTD promoted cell proliferation,inhibited endothelial inflammation by targeting mi RNA-217 and its corresponding protein-coding genes.3.β-Sitosterol demonstrated convincing endothelial protective effects similar to BBTD.And mi RNAs might be the targets of β-sitosterol and play vital roles in transcriptional regulation in endothelial protective effects against ox-LDL.The present study confirmed that Pinellia ternata is the principal drug of BBTD,β-sitosterol is one of the main effective components at the molecular level. |
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